The invention relates generally to methods and systems for processing samples on porous substrates.
Porous substrates, such as cellulose matrices (e.g. 31 ETF, FTA and FTA elute cards available from Whatman) are often used to store biological samples, such as blood. A new application area for these cards is in the pharmaceutical industry, which is using them to store dried blood samples from pharmacokinetic and toxicokinetic studies. When it is time to analyze the amount of drug or drug metabolite in the dried blood spot, the current methods require the user to cut the sample out of the card, usually a 1-6 mm diameter circle, place the cut disc in a vial or well with extraction fluid, and then shake/vortex for a set period of time. The extraction fluid is then removed and analyzed using a method such as LC-MS.
The pharmaceutical industry is expecting to process a large number of samples per day and is therefore looking for ways to automate the process. The current workflow of disc cutting and extraction, poses several problems when facing the challenge of automation. The primary problems arise from the cutting step. The small cut discs are highly prone to the effects of static electricity or even a light breeze. There are numerous reports of cut discs being lost during the cutting step or during transport of the cut discs. Cross-contamination is another significant problem associated with having to cut pieces out of the FTA cards because small fibers are often released during cutting. These small fibers can then cause cross-contamination between samples.
Previous attempts to automate the workflow include cutting out a portion of the card with sample dried on it. The cut disc is then placed in a vial/well, to which extraction fluid is added, and then shaken/vortexed for a set period of time. Alternatively, the cut disc is placed in a device that allows one to flow fluid through it to extract analytes. All of these approaches suffer from the problems and risks associated with cutting (e.g. lost sample discs, loose fiber contamination, contamination from the cutting blade).
Another approach has been to pre-cut a portion of a blank card, place the sample on the pre-cut disc of substrate, and then extract from the entire disc (by vortexing, shaking, or flow-through). Although this process addresses some of the risks associated with cutting (e.g. the cutting is done before sample application), it has limited application of use and does not allow one to analyze the sample multiple times. The uses of this method are limited because of the dependence of this process on the amount of blood fixed to the card. If the amount of sample applied to the precut disc is not consistent, the amount of drug or drug metabolite will also not be consistent. There are many situations and environments where it is difficult to achieve an accurate and consistent amount of sample collection. Inconsistency may, for example, be due to the manner in which the sample is collected (i.e. fingerstick for blood) or the training level of the people collecting the samples.
Another approach has been to place the card on a hard surface and then press down with a circular knife-edge, which presses against card but does not cut through it. Extraction buffers are then passed over the surface of the card that is isolated by the knife-edge. This method avoids cutting, but does not ensure that the fluid extracts from the full depth of the card (e.g. only the analytes at the surface may be extracted). It also does not provide a way to remove the fluid from the isolated area of the card before removing the knife-edge. This could lead to fluid wicking into the surrounding area after the knife-edge is removed. This would damage the remaining sample making re-sampling from another position on the card difficult or impossible. Also, when flowing fluid over the top of the sample, fibers are sometimes released. This approach also uses an in-line frit to remove released fibers. However, these fits may become clogged overtime and cause cross-contamination and thus must be cleaned or replaced between samples and this requires additional steps from the user.